The overall goal of this project is to improve our understanding of the mechanisms and processes underlying tumor hypoxia images obtained with nuclear methods. Towards this goal, we shall use tumor models that contain a hypoxia inducible reporter gene to allow the cascade of molecular events induced by hypoxia to be either invasively or non-invasively visualized by optical and nuclear imaging methods. Specifically, the intra-tumor distribution of the expression of the reporter gene construct HRE-tkeGFP (the fusion gene of the herpes simplex virus type 1 thymidine kinase tk and the enhanced green fluorescence protein eGFP), under the control of the hypoxia responsive element HRE will be imaged with microPET, digital autoradiography and fluorescence microscopy. Because the reporter gene is under the control of the HRE, we can determine the distribution of the initial hypoxia-induced event and provide a link between the biology of tumor hypoxia and other hypoxia-associated endpoints and surrogates. Specifically, the distributions of reporter gene expression will be compared with patterns of endogenous (e.g. HIF-1alpha and Ca9) and exogenous (e.g. pimonidazole and EF5) hypoxia-associated markers, as assessed by immunohistochemical (IHC) analysis, In addition, microPET and autoradiography images of reporter gene expression will also be compared to the images of exogenous PET hypoxia imaging agents that are under clinical evaluation: 18F-FMISO, 18F-EF5 and 124I-IAZGP. In linking hypoxia-induced molecular events to PET images, this study provides the first direct validation of non-invasive hypoxia imaging. As a reference, we shall use physical probes to perform intra-tumoral pO-2 measurements that are spatially co-registered with the images. We shall then apply these techniques to quantify the effect of interventions that modulate the effects of hypoxia including carbogen/hyperbaric oxygen breathing as well as re-oxygenation following radiation treatment. Finally, we propose to develop methods for spatial co-registration of the data from various sources: PET, autoradiography and NMR images, pO2 probe data, tumor histology and IHC analysis of endogenous or exogenous markers, with the belief that the combined datasets from complementary methods will lead to an improved assessment of tumor hypoxia.